ORIENTATION-DEPENDENT AND SEQUENCE-SPECIFIC EXPANSIONS OF CTG CAG TRINUCLEOTIDE REPEATS IN SACCHAROMYCES-CEREVISIAE/

A quantitative and selective genetic assay was developed to monitor ex pansions of trinucleotide repeats (TNRs) in yeast. A promoter containi ng 25 repeats allow's expression of a URA3 reporter gene and yields se nsitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When inte grated at either of two chromosomal loci, expansion rates were 1 X 10( -5) to 4 X 10(-5) per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant ch anges in CTG expansion rates occurred in strains deficient in the mism atch repair gene MSH2 or the recombination gene RAD52. The frequent na ture of CTG expansions suggests that the threshold number for this rep eat is below 25 in this system. In contrast, expansions of the complem entary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid wa s inverted within the chromosome, switching the leading and lagging st rands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG exp ansions, the largest gain in tract size was 38 repeats. The control re peats CTA and TAG shelved no detectable rate of expansions. The orient ation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.